Affinity-based protein profiling (AfBPP) allows us to identify target proteins that bind drugs or other small molecules of interest in complex samples. As an enrichment technique, label-free AfBPP often generates data with high missingness, particularly in negative control samples. We developed an R package, chemoprotR, which enables both quantitative and qualitative statistical analyses of chemoproteomic data, and applied it to the identification of specific benzodiazepine drug targets in the brain. Benzodiazepines comprise a class of drugs that affect GABAA receptors through positive allosteric modulation, but benzodiazepine interactions with other proteins are not fully understood. To this end, we synthesized benzodiazepine affinity-based probes (AfBPs) and applied them to rat brain synaptosomes. Our benzodiazepine AfBPs identified GABAA receptor subunits and other proteins with ion channel functions. Across the three probes, there was minimal overlap in protein targets identified by competitive labeling with flurazepam, and FR-DA, the probe based on flurazepam, yielded more significant protein targets than the probes based on flunitrazepam. These results demonstrate the ability of benzodiazepine AfBPs to identify protein targets when used with an authentic benzodiazepine to compete for binding sites and highlight the utility of combined statistical analyses for the interpretation of presence-absence data in AfBPP data sets.

Benzodiazepines (BZDs) are widely used in the treatment of psychiatric disorders, but their association with suicidal/self-injurious behavior is conflicting. This study investigated the relationship between BZDs and suicidal and self-injurious behavior, by analyzing data from the FDA Adverse Event Reporting System. We analyzed FDA Adverse Event Reporting System data from January 2004 and March 2025, to analyze adverse events (AEs) associated with suicidal and self-injurious behavior in BZDs. Thirty-eight BZDs were initially identified via the WHO Anatomical Therapeutic Chemical Code System. After excluding 26 BZDs with insufficient psychiatric disorder-related AEs (<50 reports), 12 (diazepam, chlordiazepoxide, oxazepam, potassium clorazepate, lorazepam, bromazepam, clobazam, alprazolam, flurazepam, triazolam, temazepam, and midazolam) were retained for disproportionality analysis using zolpidem as a control. Reporting odds ratios (RORs) were calculated, and subgroup analysis assessed risk variations by age and gender. Among 52,767 psychiatric disorders AEs, 9474 (17.95%) involved suicidal and self-injurious behaviors. Compared to zolpidem, diazepam (ROR = 1.38), chlordiazepoxide (ROR = 1.62), oxazepam (ROR = 2.14), lorazepam (ROR = 1.11), alprazolam (ROR = 1.64), flurazepam (ROR = 3.26) triazolam (ROR = 1.68), and temazepam (ROR = 1.72) showed significantly elevated risks, while clobazam (ROR = 0.46) and midazolam (ROR = 0.37) demonstrated protective effects. Subgroup analyses revealed higher risks in females using diazepam, oxazepam, lorazepam, alprazolam and temazepam. Adults <65 years who used potassium clorazepate, lorazepam, clobazam, alprazolam, or triazolam faced a significantly higher suicide-related risk than those ≥65 years. Compared to zolpidem, BZDs demonstrate varied suicide-related risks, which necessitated personalized risk-benefit evaluations and increased monitoring for high-risk agents such as flurazepam and alprazolam.

Dissolved black carbon (DBC) within aquatic dissolved organic matters demonstrates potent photochemical activity, yet its effects on emerging contaminant transformation and structure-reactivity relationships remain inadequately characterized. In this study, benzodiazepines were employed as model emerging contaminants to investigate the photoactivity, mediation effect, and structural-activity relationships of five DBC samples and four well-studied dissolved humic substances (DHSs) under simulated sunlight irradiation. DBC efficiently generated superoxide anions (O2[·-]), singlet oxygen ([1]O2), and triplet excited states, thereby facilitating the phototransformation of benzodiazepines. Under DBC mediation, midazolam and flurazepam achieved transformation rates of 61.1-99.5% and 19.0-84.6%, respectively, within 8 h, exceeding direct phototransformation efficiencies (1.3-13.3%). Triplet excited states were identified as the dominant reactive intermediates, contributing more than 46.7% to the photoreactions. At equivalent total organic carbon levels, DBCs averagely exhibited 1.3-, 2.7-, and 3.3-fold higher photoactivity (O2[·-], [1]O2, and triplet excited states) alongside 2.5- and 4.7-fold enhanced benzodiazepine (midazolam and flurazepam) transformation compared to DHSs based on quantum yield measurements. Phototransformation pathways of benzodiazepines via DBC-mediation included [1]O2 oxidation and charge transfer with triplet excited states, generating charge-separated intermediates that subsequently induce ring cleavage and coupling reactions. The molecular structures of DBC and DHS were characterized using UV-visible spectroscopy and Fourier transform-ion cyclotron resonance mass spectrometry. To elucidate the structure-reactivity relationship, Spearman rank correlation analysis, structural equation modeling, and orthogonal partial least squares regression were employed. The results identify molecular weight, aromaticity, and oxidation degree as key structural determinants of photoactivity. Low-molecular-weight compounds containing condensed aromatic structures and lignins demonstrate superior photoactivity in DBC/DHS structures, driving triplet excited-state and [1]O2 formation, while O2[·-] might be generated from different structures. This study elucidated DBC mediation mechanisms and structure-reactivity relationships in benzodiazepine phototransformation, identifying low-molecular-weight fractions containing condensed aromatic and lignin moieties as dominant photoactive drivers in aquatic environment.

Esta hoja trata de la exposición a la flurazepam en el embarazo y durante la lactancia. Esta información no debe usarse como un sustituto de la atención médica o los consejos de su proveedor de atención de salud.

This sheet is about exposure to flurazepam in pregnancy and while breastfeeding. This information should not take the place of medical care and advice from your healthcare provider.

Agitated depression, also known as melancholia agitata, is a variant of depression characterized by severe symptoms of psychomotor agitation, inner unrest, anxiety, restlessness, prominent vegetative symptoms, and a high risk of suicide. This form of depression is reported to worsen with antidepressants and potentially improve with the use of ECT, lithium, antiepileptics, antipsychotics, and benzodiazepines. We describe a case of a 73-year-old female with a prior history of depression and generalized anxiety disorder who was maintained on flurazepam for 44 years and was admitted for severe depression with psychomotor agitation, prominent vegetative symptoms, thought perseveration, indecisiveness, and psychotic features that emerged following the discontinuation of flurazepam. Symptoms did not resolve with the use of alternative benzodiazepines such as nitrazepam and temazepam and further worsened with the use of several antidepressants. She finally had a complete resolution of these symptoms with a combination of alprazolam, zopiclone, and olanzapine. This case provides insight into this unique variant of depression and the role of GABA agonists in its pathology and management.

Despite of being the drugs of the same therapeutic class (Benzodiazepines), each of them shows different actions prominently. It is commonly seen that Bromazepam, Clonazepam, and, Alprazolam are prescribed for the treatment of anxiety disorders, panic disorders, and phobias. On the other hand, Midazolam, Temazepam, Flurazepam, and Nitrazepam are indicated for the treatment of insomnia and Lorazepam is considered as a drug having anticonvulsant effects. As the mechanism of action is the same, there should be some differences in the binding patterns with the proteins that create differences in their impacts on the body. A deep screening of the binding patterns of the available Benzodiazepines in the market to the GABAA receptor will be beneficial to find out the responsible amino acids for being accountable for showing any specific action. This reveal will help design new molecules with the highest beneficial effect and lowest toxicity in the body. The in silico method provides the initial level of understanding regarding the binding patterns, performing in vitro and in vivo experiments will be more specific to claim the benefits of newly designed drugs.

What an honor to write about Dr. Edward O. Bixler's contributions to the sleep field. In 1967, Dr. Bixler published a case report on a chimpanzee with implanted brain electrodes while working at an Air Force base in New Mexico. A few years later, in 1971, he published on the sleep effects of flurazepam in individuals with insomnia together with Dr. Anthony Kales, data that he had collected when the Sleep Research & Treatment Center (SRTC) was housed at the University of California Los Angeles. Dr. Bixler, a meticulous scientist, learned from Dr. Kales, a devoted clinician, to study "the whole patient, and all aspects of sleep," a legacy that continued when the SRTC moved to Penn State in Hershey. Indeed, Dr. Bixler's tenure at Penn State from 1971 until 2019 kept the science of the SRTC focused on that premise and helped translate scientific evidence into clinical care. He not only contributed early to the pharmacology of sleep and the effects of hypnotics, but he was also a pioneer in "sleep epidemiology." His "Prevalence of sleep disorders in the Los Angeles metropolitan area" study of 1979 was the first rigorous epidemiological study on sleep disturbances. Starting in 1990, he established the Penn State Adult Cohort to estimate the prevalence and natural history of sleep-disordered breathing and other sleep disorders in adults. Inspired by life-course epidemiology, he established in 2001 the Penn State Child Cohort to estimate the same phenomena in children. This Living Legend paper captures and highlights Dr. Bixler's enduring legacy to sleep science.

Sleep medications often carry residual effects potentially affecting driving safety, warranting network meta-analysis (NMA). PubMed/EMBASE/TRID/Clinicaltrials.gov/WHO-ICTRP/WebOfScience were inquired for randomized controlled trials of hypnotic driving studies in persons with insomnia and healthy subjects up to 05/28/2023, considering the vehicle's standard deviation of lateral position - SDLP (Standardized Mean Difference/SMD) and driving impairment rates on the first morning (co-primary outcomes) and endpoint. Risk-of-bias, global/local inconsistencies were measured, and CINeMA was used to assess the confidence in the evidence. Of 4,805 identified records, 26 cross-over RCTs were included in the systematic review, of which 22 entered the NMA, focusing on healthy subjects only. After a single administration, most molecules paralleled the placebo, outperforming zopiclone regarding SDLP. In contrast, ramelteon 8 mg, daridorexant 100 mg, zolpidem 10 mg bedtime, zolpidem middle-of-the-night 10 mg and 20 mg, mirtazapine 15-30 mg, and triazolam 0.5 mg performed significantly worse than placebo. Lemborexant 2.5-5 mg, suvorexant 15-20 mg, and zolpidem 3.5 mg middle-of-the-night associated with lower impairment than zopiclone. Repeated administration (maximum follow-up time of ten days) caused fewer residual effects than acute ones, except for flurazepam. Heterogeneity and inconsistency were negligible. Confidence in the evidence was low/very low. Sensitivity analyses confirmed the main analyses. Most FDA-approved hypnotics overlapped placebo at in-label doses, outperforming zopiclone. Repeated administration for 15 days or less reduced residual effects, warranting further research on the matter.

Type A γ-aminobutyric acid receptors (GABAARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants[1-3]. However, our understanding of GABAAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits[4] and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAAR assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem (ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone (APG)). Using cryo-electron microscopy (cryo-EM) analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1β2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/β subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABAARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABAAR assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed.

Flurazepam is an orally available benzodiazepine used for therapy of insomnia. As with most benzodiazepines, flurazepam therapy has not been associated with serum aminotransferase or alkaline phosphatase elevations; clinically apparent liver injury from flurazepam has been reported, but is very rare.

Type A GABA receptors (GABA A Rs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anesthetics, sedatives, hypnotics, and antidepressants. However, our understanding of GABA A R pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from a total 19 different subunits and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABA A R assemblies containing the widely expressed α 1 subunit, and elucidate their structures in complex with drugs used to treat insomnia (zolpidem and flurazepam) and postpartum depression (the neurosteroid allopregnanolone). Using cryo-EM analysis and single-molecule photobleaching experiments, we uncover only three structural populations in the brain: the canonical α 1 β2γ 2 receptor containing two α 1 subunits and two unanticipated assemblies containing one α 1 and either an α 2 , α 3 or α 5 subunit. Both of the noncanonical assemblies feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, allopregnanolone is bound at the transmembrane α/β subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABA A Rs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify both the pore diameter and binding environments for GABA and insomnia medications. Together, our data reveal that GABA A R assembly is a strictly regulated process that yields a small number of structurally distinct complexes, defining a structural landscape from which subtype-specific drugs can be developed.